Production of recombinant lubricin

ABSTRACT

Disclosed are new recombinant isoforms of human-like lubricin or PRG4 glycoprotein having outstanding lubrication properties and a novel glycosylation pattern, and methods for their manufacture at high levels enabling commercial production.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. provisionalpatent application No. 61/894,366 filed Oct. 22, 2013, the entirety ofwhich is incorporated by reference herein.

FIELD OF THE INVENTION

The inventions disclosed herein relate to methods of producingcommercial quantities of compositions of matter comprising recombinanthuman-like lubricin using transfected cells. More particularly, theinventions relate to production at commercial scale of novel forms oflubricin which have excellent lubricating properties and which may beformulated and used for prophylactically or therapeutically treatingvarious conditions ranging, for example, from joint pain to dry eyedisease.

BACKGROUND OF THE INVENTION

The proteoglycan 4 (PRG4) gene encodes highly glycosylated surfacelubricating proteins named lubricin, megakaryocyte stimulating factor(MSF), or superficial zone protein (SZP). (See Jay, Curr. Opin. Orthop.15, 355 (2004); U.S. Pat. No. 6,743,774; U.S. Pat. No. 6,960,562).Lubricin is expressed from the PRG4 gene (SEQ ID NO: 2) with a fulllength spanning 12 exons, although multiple, naturally occurringtruncated versions have been reported. A large “mucin like” centraldomain of 940 amino acids (encoded by exon 6) comprises some 70+KEPAPTT-like sequences and is glycosylated heavily. The glycoproteincomprises core 2 glycosylation residues and a multiplicity of core 1glycans (O-linked β(1-3) Gal-GalNAc oligosaccharides), at least thelatter of which have been shown to mediate its primary physiologicalfunction, boundary lubrication (Jay et al., Glycoconj J 18, 807 (2001)).PRG4 has been shown to be present at the surface of cartilage, synovium,tendon, and meniscus, in the tear film and at other anatomical sites.PRG4 has been shown to contribute to the boundary lubrication ofapposing articular cartilage surfaces. PRG4 has been shown to exist notonly as a monomer but also as a dimer and multimer disulfide-bondedthrough the conserved cysteine-rich domains at both N- and C-termini,(Schmidt et al., Biochim Biophys Acta. 1790(5):375-84 (2009); Kooyman etal., Paper No. 255, 56^(th) Ann. Meet of Orthop. Res. Soc., 2010).

At the cartilage interface of synovial joints there are at least twophysicochemical modes of lubrication in action. These have beenclassified as “fluid film” and “boundary.” The operative lubricationmodes depend on the normal and tangential forces on the articulatingtissues, on the relative rate of tangential motion between thesesurfaces, and on the time history of both loading and motion. Thefriction coefficient, μ (a dimensionless unit, ratio of the measuredfrictional force between two contacting surfaces in relative motion tothe applied normal force), provides a quantitative measure oflubrication.

One type of fluid-mediated lubrication or “fluid film” mode ishydrostatic. At the onset of loading and typically for a prolongedduration, the interstitial fluid within cartilage becomes pressurized,due to the biphasic nature of the tissue, fluid may also be forced intothe asperities between articular surfaces through a weeping mechanism.Pressurized interstitial fluid and trapped lubricant pools comprisinghyaluronic acid may therefore contribute significantly to the bearing ofnormal load with little resistance to shear force, facilitating a verylow friction coefficient. Also, at the onset of loading and/or motion,squeeze film, hydrodynamic, and elastohydrodynamic types of fluid filmlubrication may occur, with pressurization, motion, and deformationacting to drive viscous lubricant from and/or through the gap betweentwo surfaces in relative motion.

In boundary lubrication, load is supported by surface-to-surfacecontact, and the associated frictional properties are determined bylubricant surface molecules, i.e., lubricin species. This mode isimportant because the opposing cartilage layers make contact over +/−10%of the total area via interlocking, flattened asperities, and thislikely is where most of the friction occurs. Boundary lubrication, inessence, mitigates “stick-slip” (Meyer et al., Nanoscience: Friction andRheology on the Nanometer Scale, World Scientific Publishing Co. Pte.Ltd, River Edge, N.J., (2002), pp. 373), that is, spontaneous jerkingmotion that can occur while interfacing weight bearing cartilagesurfaces are sliding over each other, and is therefore manifest asdecreased resistance both to steady motion and the start-up of motion.Typical wear patterns of cartilage surfaces suggest that boundarylubrication of articular cartilage is critical to the protection andmaintenance of the articular surface structure. For example, lubricinnull mice show wear but newborn nice, which are not weight bearing, donot. (Jay et al., Arthritis and Rheumatism, 56:3662-3669 (2007).

With increasing loading time and dissipation of hydrostatic pressure,lubricant-coated surfaces bear an increasingly higher portion of theload relative to pressurized fluid, and consequently, μ can becomeincreasingly dominated by the boundary mode of lubrication. A boundarymode of lubrication is therefore indicated by a friction coefficientduring steady sliding being invariant with factors that influenceformation of a fluid film, such as relative sliding velocity and axialload. For articular cartilage, it has been concluded that boundarylubrication is certain to occur, although complemented by fluidpressurization and other mechanisms. The lubrication mechanism at theinterface of the cornea and eyelid during the eye blink does not involvea significant load, accordingly easing the physicochemical requirementsfor effective lubrication, and therefore is likely quite different fromcartilage lubrication. However, it has been proposed that a boundarymode of lubrication can become dominant when tear film is compromised,such as in dry eye disease.

The two mechanical components of synovial fluid thought to beresponsible for its remarkable lubrication properties are lubricin andhyaluronic acid (or hyaluronate or “HA”, hereinafter usedinterchangeably). Lubricin has been shown to function as a boundarylubricant in articulating joints and to protect cartilaginous surfacesagainst frictional forces, cell adhesion and protein deposition. Forexample, U.S. Pat. Nos. 6,960,562 and 6,743,774 disclose a lubricatingpolypeptide comprising substantially pure PRG4 isoforms, and methods oflubricating joints or other tissues by administering systemically ordirectly to tissues. HA per se has been shown to decrease μ over saline(0.12 in 3.3 mg/ml HA vs. ˜0.24 in PBS) at a cartilage-cartilageinterface under boundary mode lubrication, and lubricin alone decrease μto still lower levels, but synovial fluid comprising HA in combinationwith lubricin can impart to interfacing surfaces a coefficient offriction not achieved by lubricin alone or by synthetic mixtures of HAand lubricin. No synthetic composition of lubricin and HA has yet beenable to fully duplicate the low coefficient of friction imparted bynative form synovial fluid. HA from various sources and variousmolecular weights have been tested in admixture with lubricins expressedin vitro from synoviocytes, bovine lubricins, lubricins extracted fromsynovial fluid and “reconstituted” in HA, and lubricins expressed inmicrogram quantities in early efforts to make it using recombinant DNAtechnology.

Previous attempts at recombinant production of full length lubricin at ascale suitable for commercial exploitation have not been successful. Thevery low, single or double-digit milligram per liter rate of productionof human lubricin species expressed from CHO cells is considered too lowto support a commercial product. One approach to solving this problemwas to truncate the number of repeats in exon six, and therefore reducethe mass of glycosylation side chains while retaining at least somelubricating ability (see, e.g., U.S. Pat. Nos. 7,642,236 and 7,893,029).This approach reportedly resulted in a gross productivity (beforepurification) of the truncated construct of three to four hundredmilligrams per liter.

SUMMARY OF THE INVENTION

It has now been discovered that the human PRG4 gene can be used toproduce large, commercial quantities of a novel, highly glycosylatedhuman-like form of lubricin, hereinafter referred to simply as“lubricin,” multimeric lubricin, rhlubricin, or rhPRG4. This isaccomplished as disclosed herein by transfecting the human PRG4 gene(hPRG4) into certain modified Chinese hamster ovary (CHO) cells whichhave been discovered to be competent to post-translationally glycosylateexpressed proteins on a large scale, and then culturing the cells incommercial scale volumes of media, for example, at least 10 liters, moretypically at least 50 liters, preferably at least 100 liters or at least500 liters, and most preferably at 1,000 liters or more.

The lubricin of this invention comprises polydisperse lubricin monomerunits forming dimers and multimers and optionally free monomers. Eachunit is heavily and variably glycosylated, with the glycosidic residueside chains contributing at least 30%, often 35% or 40%, and possibly ashigh as 45% or more of its molecular weight.

In native human lubricin, glycosylations consist of core 1 O-linkedGalNAc-Gal (N-acetylgalactosamine-galactose) disaccharide, at least 60%of which is terminally substituted with a sialic acid, and also core 2glycosylation involving addition to core 1 of GlcNac(N-acetylglucosamine) monosaccharides in various isomericconfigurations. (See, e.g., Estrella et al., Biochem J., 429(2):359-67(2010)).

The recombinant material produced as disclosed herein is enriched incore 1 glycans, as compared to native form human lubricin. Itsglycosylation comprises at least 95% core 1 side chains, more likelymore than 98% or 99%. Furthermore, the side chains often are sulfated toa degree not seen in native human lubricin. This distinguishes therhPRG4 of this invention from native hPRG4. The increased sulfationcontent is believed to add additional negative charges to the mucinousglycoprotein which may serve to enhance its ability to repel nearbybiomolecules and thus increase its lubricity, and to stiffen themolecular structure, making it more rigid from a molecular standpoint,which can assist in its ability to function in reducing nanoscale andmesoscale friction.

The full length (non-truncated) lubricin monomer sequence (SEQ ID NO:1)comprises 1404 amino acids, or approximately 151 kDa in core protein.The signal sequence of human lubricin is residues 1-24 of SEQ ID NO:1.Accordingly, the mature form of human lubricin is residues 25-1404 ofSEQ ID NO:1. Exhaustive reduction of the recombinant product produced asdisclosed herein yields a monomeric species with an apparent molecularweight of about 300 kDa-460 kDa, as estimated by comparison to molecularweight standards in a number of molecular weight determinationtechniques including SDS tris-acetate 3-8% polyacrylamide gelelectrophoresis. Glycosylation analysis using mass spectrometrytechniques and other work in combination suggested that the truemolecular weight of a glycosylated recombinant monomer (as opposed toinferred from gel mobility) likely is in the range of 220-280 kDa, andis unlikely to exceed about 300 kDa. From a total of about 329 possibleO-linkages (284 of which are threonines) potentially available as sitesof O-linked glycosylation in the sequence of the lubricin monomer, avarying and unknown large number are substituted (100 to 150, perhaps ashigh as 200 or 220). Of the total glycosylation, about half comprise twosugar units (GalNAc-Gal), and half three sugar units (GalNAc-Gal-Sialicacid). The most abundant form is sulfated Gal-GalNac, the next mostabundant is sialylated Gal-GalNac.

The lubricin expression product is resistant, although not immune, tobreakdown into monomeric or dimeric lubricin species. The results ofexhaustive reduction imply that it comprises disulfide cross-linksbetween and within monomer units. Also, treatment with denaturingbuffers without reduction can result in lower molecular weight products,suggesting higher order quaternary structures where the chains also areheld together by hydrophobic interaction, hydrogen bonding, physicalentanglement and/or other non-covalent associations allowing forself-assembly. The dimers and multimers are polydisperse. Molecularspecies within it typically have molecular weights of at least about450-600 kDa, and multimeric species frequently 2,000 kDa or more.Typically, some species of the non-reduced complex essentially do notenter the 3-8% SDS-PAGE gel in an electrophoresis experiment. The largerspecies of the complex is believed to comprise between three and five,and perhaps as many as 20 monomer units.

Without wishing to be bound by theory, it is believed such largersupramolecular components are formed as a function of monomer/dimerconcentration. Currently, a concentration of at least about 0.5 mg/mlmonomer/dimer is believed to be optimal for spontaneous formation oflarger complex. Concentrations well below this, e.g., less than about0.1 mg/ml, comprise monomers and dimers, and only a minor amount ofcomplex; concentrations well above can form aggregates visible with thenaked eye as a cloudy or hazy solution. Surfactants, preferablyphysiologically compatible nonionic surfactants that are generallyregarded as safe, e.g., polyoxyethylene-based surfactants, or excipientsmay be used to prevent large aggregate formation while permittingformation of the complex, which appears always to be present togetherwith dimeric species.

Testing of preparations comprising the lubricin of the invention showsthat its lubricating and tissue protection properties under load mayexceed that of recombinant lubricin heretofore known in the art. Withoutwishing to be bound by theory, the inventors hereof hypothesize thatwhile stick-slip phenomenon occurs during movement of unlubricatedinterfacing tissues under load, a coating of the lubricin of theinvention can transfer shear away from the underlying cartilage surfaceto layers within the coating of polydisperse lubricin. That is, theinventors believe that under load and reciprocating motion, theunderlying surface experiences less shear, preserving its integrity, asmolecules of lubricin within the coating slip over one another, thenlikely rearrange when the load is removed. (See, e.g., Lee et al., PNAS,110(7):E567-574 (2013)). The authors state that joint wear is notdirectly related to the friction coefficient, but more directly relatedto stick-slip sliding, and that the different molecular components ofthe joint work synergistically to prevent wear.

In any event, the lubricin product of the invention, when tested,exhibits outstanding lubricating properties, resulting in coefficientsof friction (both static and dynamic) often within 150%, 120%, 110% oressentially equal to the coefficients of purified, native bovinelubricin, as measured by cartilage-on-cartilage lubrication testing asdisclosed herein. In these tests, the human-like glycoprotein of theinvention achieves static coefficients of friction at or below 0.5 andlower than 0.2 (depending on test conditions as disclosed herein), andkinetic coefficients of friction often at or below about 0.1, both asmeasured by depressurized cartilage upon cartilage bearings in vitro,with a stationary area of contact. When combined with hyaluronic acid(HA), these values improve to below about 0.3 and less than 0.1 for thecertain static measurement (depending on dwell time) and less than 0.1for the kinetic measurement, quite close to the accepted value forsynovial fluid. Accordingly, such compositions can dramatically reducejoint wear.

Accordingly, one aspect of the invention comprises a method for thecommercial production of lubricin. In one embodiment, the methodincludes the steps of culturing, in a medium, Chinese hamster ovary(CHO) cells transfected with and which express the human PRG4 gene andpost-translationally glycosylate the expression product for a time andunder culture conditions sufficient to produce a lubricin glycoprotein,and purifying the lubricin glycoprotein from said medium. For example,in some embodiments, the lubricin glycoprotein is separated from hostcell proteins and other contaminants in the extracellular broth to atleast partially purify it. The recombinant protein need only be enrichedfrom the culture medium, rather than purified to homogeneity in order tobe purified for the purposes of the method of the invention. The methodis sufficient to produce a lubricin glycoprotein having at least 30% byweight glycosidic residues at a concentration in the medium of at least0.4 g/L.

In some embodiments, the CHO cells are CHO-M cells comprising a nucleicacid encoding the human PRG4 gene. In other embodiments the CHO cellsare transfected with a first vector comprising a nucleic acid encoding achromatin element and are transfected with a second vector comprising anucleic acid encoding the human PRG4 gene. The chromatin element may bea boundary element, a matrix attachment region, a locus control region,or a universal chromatin opening element. In a preferred embodiment, thechromatin element is a matrix attachment region.

In yet another embodiments, the CHO cells are transfected with a firstvector comprising a nucleic acid encoding a chromatin element andencoding the human PRG4 gene and are transfected with a second vectorcomprising a nucleic acid encoding a chromatin elements and encoding thehuman PRG4 gene. In a preferred embodiment, the chromatin elements inthe first and second vectors are matrix attachment region.

In some embodiments, at least 30%, at least 35%, at least 40%, or atleast 45% of the weight of the dimeric or multimeric lubricinglycoprotein is the weight of glycosidic residues. In some embodiments,greater than 30%, greater than 35%, greater than 40%, or greater than45% of the weight of the dimeric or multimeric lubricin glycoprotein isthe weight of glycosidic residues. The glycosidic residues may differfrom those of native human lubricin as the glycosylation of therecombinant human-like lubricin is at least 90%, at least 95%, or atleast 99% by weight core 1 glycosylation. Also, in some embodiments, theglycosidic residues are enriched in sulfated monosaccharides as comparedwith native human lubricin.

The process, unexpectedly, is capable of producing commercially viablequantities of the full length lubricin glycoprotein. For example, thecells can be cultured for a time and under culture conditions sufficientto produce lubricin glycoprotein concentrations in a culture medium ofat least about 0.4 grams or 0.5 grams recombinant lubricin per liter,preferably at least 0.8 grams per liter, and most preferably at least1.0 grams of lubricin per liter of culture medium in a culture, forexample, of at least about 10, 50 or 100 liters. The process whenoptimized may produce as much as 2.0, at least 2.5, or at least 3.0grams of lubricin per liter of culture. Depending on development of anoptimized purification protocol, it will be possible to obtain at leastabout 200 milligrams of purified recombinant lubricin per liter,preferably at least 300 mg/L, more preferably at least 500 mg/L, andmost preferably more. As far as applicants are aware, these levels ofproductivity have never before been achieved in recombinant expressionof any mucin-like protein, or a protein comparable in size to lubricin,and no previous attempt at expression of PRG4 has succeeded in producingmaterial having the properties of the product described herein.

In preferred embodiments, monomeric lubricin species often areco-purified from the culture medium in admixture with the multimericprotein species. The multimeric species are rich in dimeric lubricinspecies. For example, in some embodiments, the method produces a mixtureof recombinant lubricin that includes monomeric, dimeric, and multimericlubricin species. In some embodiments, the lubricin glycoproteincomprises at least five disulfide-bonded or non-covalently associatedindividual glycosylated amino acid chains and has a molecular weight ofat least 1200 kDa.

The glycoprotein produced according to the methods of the invention,when tested using the protocol outlined below, produces a coefficient offriction approaching the lowest values ever observed for purified nativemammalian lubricin. For example, in some embodiments, the recombinantlubricin glycoprotein is a multimeric protein that produces a staticcoefficient of friction no greater than 150% of the static coefficientof friction of purified native bovine lubricin as measured in acartilage on cartilage friction test. In other embodiments, therecombinant lubricin glycoprotein is a multimeric protein that producesa static coefficient of friction no greater than 120% of the staticcoefficient of friction of purified native bovine lubricin as measuredin a cartilage on cartilage friction test. In yet other embodiments, therecombinant lubricin glycoprotein is a multimeric protein that producesa static coefficient of friction no greater than 110% of the staticcoefficient of friction of purified native bovine lubricin as measuredin a cartilage on cartilage friction test.

Another aspect of the invention is directed to compositions of arecombinant, multimeric, lubricin glycoprotein expressed from the humanPRG4 gene in a host cell culture. The recombinant lubricin glycoproteinis at least 30% by weight glycosidic residues and produces a dynamiccoefficient of friction no greater than 150% of the dynamic coefficientof friction of purified native bovine lubricin as measured in acartilage on cartilage friction test.

In some embodiments, the recombinant lubricin glycoprotein is at least35%, at least 40% or at least 45% by weight glycosidic residues.

In some embodiments, the recombinant lubricin glycoprotein produces adynamic coefficient of friction no greater than 110% or no greater than120% of the dynamic coefficient of friction of purified native bovinelubricin as measured in a cartilage on cartilage friction test.

In some embodiments, the glycosidic residues of the recombinant lubricinmay differ from those of native human lubricin as the glycosylation ofthe recombinant lubricin is at least 90%, at least 95%, or at least 99%by weight core 1 glycosylation. Also, in some embodiments, theglycosidic residues of the recombinant lubricin are enriched in sulfatedmonosaccharides as compared with native human lubricin.

In some embodiments, the recombinant lubricin is a mixture of monomeric,dimeric and multimeric species. In some embodiments, the lubricinincludes monomeric species. In some embodiments, the lubricin includesdimeric species. In some embodiments, the lubricin includes multimericspecies. In some embodiments, the lubricin is a mixture of multimericand monomeric species.

In some embodiments, the lubricin glycoprotein comprises at least fivedisulfide-bonded or non-covalently associated individual glycosylatedamino acid chains and has a molecular weight of at least 1200 kDa.

In some embodiments, the composition of recombinant lubricinglycoprotein further includes hyaluronic acid or a salt thereof inadmixture with the lubricin.

In another embodiment, the invention is directed to a compositioncomprising a solution comprising 100 grams of human lubricin where theglycosylation of the lubricin is at least 99% by weight core 1glycosylation. In one embodiment, the lubricin is recombinant humanlubricin. In yet another embodiment, the concentration of lubricin inthe solution is at least 0.5 g/L. In yet another embodiment, thesolution is a cell culture medium.

Compositions of the invention may be used for the preparation of amedicament for any known or hereafter discovered medical or other use ofPRG4 glycoprotein, including as a coating for various devices intendedfor contact with the body (see, e.g., U.S. Patent ApplicationPublication Nos. 2009/0068247 and 2011/0142908); for the treatment of anarticular joint in a human or animal by enhancement of joint lubrication(U.S. Patent Application Publication No. 2004/0229804) orviscosupplementation (U.S. Patent Application Publication No.2008/0287369); for topical application to a tissue surface, e.g., duringsurgery to inhibit subsequent formation of adhesions or fibroticconnective tissue (U.S. Patent Application Publication No.2004/0229804); for the treatment of dry eye disease (U.S. PatentApplication Publication No. 2011/0059902); for treatment of dry mouthdisease (U.S. Patent Application Publication No. 2013/0039865); fortreatment of interstitial cystitis (U.S. Patent Application PublicationNo. 2012/0321693); as a vaginal lubricant (U.S. Patent ApplicationPublication No. 2012/0052077); for a contact lens care and storagesolution (U.S. Patent Application Publication No. 2012/0321611) or forsystemic injection to, for example, inhibit cell-cell adhesions ormotility within the vasculature (see, e.g., U.S. provisional application61/908,959 filed Nov. 26, 2013).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are plasmid maps of vectors used in development of thelubricin-expressing CHO-M clone used in the process of the invention andencoding the full length sequence of hPRG4.

FIGS. 3 and 4 depict polyacrylamide gels useful in assessing thestructure of the rhlubricin of the invention.

FIG. 5 is a graphical depiction of the productivity of one lubricinproduction run measuring micrograms of lubricin produced over time perliter of culture of transfected CHO-M cells. There are three bars ateach date of harvest. The bar at the left is “Std Curve 24 Jun 14.” Thebar in the middle is “Std Curve 24 Jul 14,” and the bar at the right is“Std Curve 25 Jul 14.”

FIG. 6 is a diagram showing core 1 glycans of rhlubricin of theinvention.

FIG. 7 is a chromatograph, with peaks labeled, showing the relativeabundance of the various di- and tri-saccharides pendent from SER andTHR residues in the rhlubricin of the invention.

FIG. 8 is a diagram showing the larger range of glycans extending intothe core 2 structures on native lubricin extracted from human synovialfluid.

FIG. 9 is a chromatograph, with peaks labeled, showing the relativeabundance of the various sugar residues in native human lubricin.

FIGS. 10A, 10B, and 10C are plots of surface tension vs. rhPRG4 and/orpolyoxyethylene surfactant concentrations showing the reduction insurface tension with increased concentration of rhPRG4.

FIGS. 11A and 11B depict data comparing the static (FIG. 11A) andkinetic (FIG. 11B) friction coefficients of rhPRG4 solution to purifiednative bovine PRG4, saline (PBS), and bovine synovial fluid, with bothPRG4 preparations 450 μg/mL. The designations a, b, and c signifystatistically significant differences in the results (p<0.05). n=7.There was no statistically significant difference in the results forrh-PRG4 (recombinant) and nPRG4 (native) as indicated by the presence of“b” above each bar in FIG. 11B.

FIGS. 12A-B depict data comparing the static (FIG. 12A) and kinetic(FIG. 12B) friction coefficients of an HA plus rhPRG4 solution tosaline, rhPRG4 alone, and bovine synovial fluid with rhPRG4 at 450 μg/mLand HA (1.5 MDa) 3.33 mg/mL. The designations a, b, c, and d above eachbar in FIG. 12B signify statistically significant differences in theresults (p<0.05), n=4.

FIG. 13 depicts data showing reestablishment of lubricity at interfacingbovine tissue surfaces after digestion of native bovine PRG4 andapplication of rhPRG4.

FIGS. 14A-D depict data showing the effect of rhPRG4 on boundarylubrication at a human cornea-eyelid interface (FIG. 14A—static, FIG.14B—kinetic) and human cornea-PDMS (FIG. 14C—static, FIG. 14D—kinetic)interfaces, of native bovine PRG4 and rhPRG4 at 300 μg/ml in saline, andsaline alone. Values are mean±SEM (n=6) with an average normal stress of14.1±2.2 and 16.9±5.3 (mean±SD) for the cornea-eyelid (AB) andcornea-PDMS (CD) interfaces, respectively. These data illustrate thevirtually identical lubricating properties of rhPRG4 and purified nativePRG4 in low load lubrication tasks.

FIG. 15 is the amino acid sequence of full length human lubricin whichis 1404 amino acids in length. The signal sequence residues (1-24) areshown in bold.

FIG. 16 is the nucleic acid sequence encoding full length humanlubricin.

DETAILED DESCRIPTION OF THE INVENTION

The inventors hereof investigated options for the production of theknown human lubricin glycoprotein using recombinant DNA techniques, withthe goal of generating a production process involving suspension cultureexploiting mammalian cells in serum-free growth medium. Unlike anyprevious effort known to applicants to produce proteins usingrecombinant DNA techniques, the challenge was to produce commercialquantities of a complex, large biopolymer who's value lay in itsnanoscale mechanical properties, as opposed to its biochemicalproperties, and those physical properties were dependent on successfulexploitation of post translational glycosylation events at a scale neverbefore observed in an engineered cell.

Previous attempts at recombinant production of full length lubricin hadyielded only low milligram per liter quantities, and a method ofproducing at least about one to two grams per liter was needed. A reviewof the literature revealed no reports of successful recombinantproduction at commercial scale of full length, properly glycosylatedlubricin, nor commercial scale expression of any mucin or mucin-likeprotein. The search did reveal reports suggesting such a highlyglycosylated glycoprotein as lubricin was quite difficult to express.See, e.g., U.S. Pat. No. 7,642,236 which states: “In order to optimizeexpression parameters and investigate the functional necessity of allapproximately 76-78 KEPAPTT-similar sequences, lubricin expressionconstructs were designed which enabled the synthesis of recombinantlubricin proteins with varying degrees of O-linked oligosaccharidesubstitution.” Productivity data of the recombinant cell linesexpressing the truncated lubricin constructs were not disclosed in thepatent.

The inventors sought out and ultimately retained Selexis S.A. of Geneva,Switzerland to produce lubricin-expressing clonal cultures, based inpart on the reported ability of the Selexis technology, involvingexpression of epigenetic regulators, to enhance production of difficultto express proteins. (See Selexis U.S. Pat. Nos. 7,129,062 and 8,252,917and U.S. Patent Application Publication Nos. 2011/0061117, 2012/0231449and 2013/0143264, the disclosures of which are incorporated herein byreference; Girod et al., Nat Methods 4(9):747-53 (2007); Harraghy etal., Curr Gene Ther. 8(5):353-66 (2008)).

Application of the Selexis technology resulted in development of clonessuccessfully expressing lubricin. After analysis, scale up andpurification, it was discovered that the newly developed recombinantproduction procedures resulted in a never before described, multimeric,heavily and differently glycosylated forms of human-like lubricin, andyields that were at unprecedented levels for such heavily glycosylated,high molecular weight, mucin-like glycoproteins. Testing of preparationsrich in the new recombinant lubricin form demonstrated unexpectedproperties and enabled production of improved physiologically compatibletissue lubricating compositions.

The rhlubricin Manufacturing Process Host Cells

The Selexis clone production work was done using its proprietary CHO-Mcell line, which contains DNA-based elements that control the dynamicorganization of chromatin, so-called matrix attachment regions. TheCHO-M cell line is a Chinese Hamster Ovary cell line derived from CHO-K1cells (ATCC, Cat. #CCL-61, Lot. 4765275) adapted to serum freecultivation conditions and used for the production of recombinantproteins. See Girod et al., Nat Methods 4(9):747-53 (2007) and theSelexis U.S. patents and publications identified above relating tomatrix attachment regions (MARs) for methods for use of MARs for thedevelopment of stable high expressing eukaryotic cell lines such as CHO,and to cells transfected to express proteins involved in translocationof expression products across the ER membrane and/or secretion acrossthe cytoplasmic membrane. CHO-M is used for the production oftherapeutic recombinant proteins and allows for higher and more stableexpression. Its use permitted isolation of clones exhibiting thedesired, high-level expression for use in production of recombinantproteins.

Matrix attachment regions (“MARs”) are DNA sequences that bind isolatednuclear scaffolds or nuclear matrices in vitro with high affinity (Hartet al., Curr Opin Genet Dev, 8(5):519-25 (1998). As such, they maydefine boundaries of independent chromatin domains, such that only theencompassing cis-regulatory elements control the expression of the geneswithin the domain. MAR sequences have been shown to interact withenhancers to increase local chromatin accessibility (Jenuwein et al.,Nature, 385: 269-272 (1997)), and can enhance expression of heterologousgenes in cell culture lines. Co-transfection of a plasmid bearing thechicken lysozyme 5′ MAR element with one or more expression vectorsresults in increased stable transgene expression which was shown toproduce a 20-fold increase in expression as compared to controlconstruct.

MARs are one type of “chromatin element” (also referred to herein asSelexis Genetic Elements or SGEs) that are disclosed in the Selexisapplications and publications referenced herein. Chromatin elements orSGEs are used to prevent the chromatin surrounding the site ofintegration of a heterologous gene into a host's chromosome frominfluencing the expression level of the incorporated gene. Chromatinelements include boundary elements or insulator elements (BEs), matrixattachment regions (MARs), locus control regions (LCRs), and universalor ubiquitous chromatin opening elements (UCOEs). SGEs shape thechromatin once the expression vector has integrated in the host cellchromosome and thus maintain the transgene in a highly transcriptionallyactive state.

The CHO-M host cells were cultivated in SFM4CHO medium (HyClone),supplemented with 8 mM L-Glutamine, hypoxanthine and thymidine (1× HT,Invitrogen). Cells were maintained under agitation (120 rpm, 25 mmstroke) in a humidified incubator at 37° C. and 5% CO2.

Vector Construction

The PRG4 gene encoding the full length 1404 AA human lubricin protein(SEQ ID NO:2) was inserted into plasmid vectors commercially availableand proprietary to Selexis S.A. (Geneva, Switzerland) for enhanced geneexpression in mammalian cells. Another sequence encoding full lengthhuman lubricin is available under GenBank Accession No. NM_005807.3.

Two expression vectors were constructed. The lubricin gene was clonedinto expression vectors carrying puromycin resistance and anothercarrying hygromycin resistance. The vector including the puromycinresistance was designated pSVpuro_C+_EF1alpha(KOZAK-ext9) EGFP_BGHpA>X_S29(2*HindIII, SalI filled) (Mw=9861). The vector including thehygromycin resistance was designated pSVhygro_C+_EF1alpha(KOZAK-ext9)EGFP_BGH pA>X_29(2*HindIII, SalI filled) (Mw=10299). The expressionvectors contained the bacterial beta-lactamase gene from Transposon Tn3(AmpR), conferring ampicillin resistance, and the bacterial ColE1 originof replication. As derivatives of pGL3 Control (Promega), the terminatorregion of the expression vectors contained a SV40 enhancer positioneddownstream the BGH polyadenylation signal. Each vector also included onehuman X_29SGE downstream of the expression cassette and an integratedpuromycin or hygromycin resistance gene under the control of the SV40promoter. X_29SGE refers to a Selexis Genetic Element (“SGE”), in thiscase a matrix attachment region (MAR), that are disclosed in the Selexisapplications and publications referenced herein Both expression vectorsencoded the gene of interest (PRG4) under the control of the hEF-1-alphapromoter coupled to a CMV enhancer. Plasmids were verified bysequencing.

Plasmid maps of the vector carrying the puromycin resistance gene andcarrying the hygromycin resistance gene are shown in FIG. 1 and FIG. 2,respectively.

Transfection

The cells were transfected by microporation using a MicroPorator™(NanoEnTek Inc., Korea) defining the pulse conditions for CHO-M cells(1250V, 20 ms and 3 pulses). Transfection efficiency was controlledusing a GFP expressing vector in parallel and showed transfectionefficiency between 50-70%. The CHO-M cells were first transfected withthe puromycin PRG4 expression vector, and stably transfected cells wereselected first by culturing on a medium containing puromycin. Moreparticularly, dilutions were dispensed onto 96-well plates, fed withinthe following week by adding 100 μL of fresh selection medium to allwells (SFM4CHO medium supplemented with 8 mM L-Glutamine, 1× HTincluding 5 μg/mL of puromycin). Twenty seven minipools were reset to24-well plates 15 days after plating by transferring the complete cellsuspension out of the corresponding 96-well into one well of a 24-wellplate primed with the same medium. Within four days 24-well supernatantswere analyzed and 14 minipools were transferred to 6-well plates (1 mLcell suspension+2 mL fresh growth medium incl. selection). Eight bestexpressing minipools were expanded three days later by suspension andcollection in spin tubes (5 mL working volume) and three days latercultivated in shake flasks (20 mL working volume). One subsequentpassage was performed before banking.

The pools of resistant cells were expanded in shake flasks to generatematerial needed for preliminary studies (1-2 mg total). Cell-free mediasamples were acquired by centrifugation of cell culture at 800 g for 5min. The expression of recombinant PRG4 was assayed by dot blotanalysis. Ten microliters of cell-free media (concentrated sample) wasapplied on a PVDF membrane (Millipore) and the samples were allowed tospot dry. A PRG4 standard was created by serially diluting PRG4 at 80μg/ml down to 2.5 μg/ml. Recombinant PRG4 was detected by means of apolyclonal antibody directed against a lubricin synthetic peptide ofPRG4 (Pierce).

Cells from the best performing minipools were next super transfected(additional transfection of already selected minipool population), usingthe second selection marker, the hygromycin resistance cassette. Thesame transfection protocol was used as described above. One day afterthis second transfection, selection was started in SFM4CHO medium, againsupplemented with 8 mM L-Glutamine and 1× HT, but including 1000 μg/mLof hygromycin. After a media exchange, within four days the three poolswere transferred to 6-well plates; all three (3) pools were expanded tospin tubes (5 mL working volume) four days later and to shake flasks (20mL working volume) within three days.

Clone Generation

The supertransfected pools then were cultivated and analyzed for growthpotential in multiple and serial experiments in an attempt to maximizecell properties.

In the first experiment, three super transfected pools (designatedP01ST, P05ST and P14ST) were transferred to 6-well plates after themedium exchange at the concentration of 100 cells/mL (2 plates for eachpool), in semi-solid medium (2× SFM4CHO medium (HyClone) and CloneMatrix(Genetics), including 8 mM L-Glutamine, 1× HT and Cell Boost 5™(HyClone), (without selection). Plated cells were screened 16 dayslater, (ClonePix™ system (Molecular Devices)) and 22 candidates werepicked and transferred to 96-well plates with growth medium describedabove (but without selection). All 18 growing candidates were reset to24-well plates six days later, by transferring the complete cellsuspension out of the corresponding 96-well into one well of a 24-wellplate (primed with 1 mL of medium). Within three days 24-wellsupernatants were analyzed and 12 candidates were transferred to 6-wellplates (1 mL cell suspension+2 mL fresh growth medium includingselection). The seven best expressing candidates were expanded five dayslater to suspension cultivation in spin tubes (5 mL working volume) inmedium (without selection) and within five days in shake flasks (20 mLworking volume).

All cell lines were banked. The performance of the three best candidateswas compared in shake flasks (seeding 3×10⁵ cells/mL, 20 mL culturevolume) within fed-batch cultivation (feed strategy—16% of originalvolume CBS solution (HyClone), 52 mg/mL, fed at day 0, 3, 4, 5, 6, 7).By day 8, the cultures contained 4.22×10⁶ to 4.95×10⁶ cells/mL and 94%to 96% viability. Cell populations of these pools were counted anddiluted for single cell plating (concentration 1 cell/well, two plates).Single colonies were fed by adding 100 μl growth medium per well after11 days (without selection). After 17 days, 99 clones were reset to24-well plates by transferring the complete cell suspension out of thecorresponding 96-well into one well of a 24-well plate (primed with 1 mLof medium). Within four days 24 were transferred to 6-well plates (3 mLfresh growth medium incl. selection). Eight clones were expanded tosuspension cultivation in spin tubes (5 mL working volume) after fourdays and all eight clones were expanded to shake flasks (20 mL workingvolume) after one medium exchange (SFM4CHO medium, supplemented with 8mM L-Glutamine and 1× HT). One subsequent passage was performed beforebanking of all candidates.

Comparison of performance of the five best candidates was done in shakeflasks (seeding 3×10⁵ cells/mL, 20 mL culture volume) with fed-batchcultivation (feed strategy A 16% of original volume CBS solution(HyClone), 52 mg/mL, fed at day 0, 3, 4, 5, 6, 7). On day three the cellnumbers in the respective cultures ranged from 1.61×10⁶ to 3.46×10⁶cells/mL with doubling times ranging from 19.8 to 30.7 hours. On day 8,the cell concentrations ranged from 4.02×10⁶ to 9.48×10⁶ cells/mL withcell viability ranging from 88.6% to 97.7%.

In the second experiment, three different super transfected pools(designated P14STcp08, P05ST11 and P14ST33) were treated to the sameprocedure as outlined above. This resulted in four clonal cell lines.Again, the performance of these clones was compared in shake flasks,resulting in day 8 cell concentrations ranging from 3.5×10⁶ to 9.48×10⁶cells/mL and viability between 75.3% and 88.1%.

A clone from the first round of ClonePix™ system selection describedabove (P14ST15) which exhibited on day eight 6.03×10⁶ cells/mL and 95.5%viability was thawed in a shake flask (20 mL working volume). Thecandidate was transferred to a single plate after one subsequentpassage, at the concentration of 200 cells/mL (1 plate) in thesemi-solid medium described above plus CloneMatrix, including 8 mML-Glutamine, 1× HT and Cell Boost 5™, without selection. Plated cellswere screened using the ClonePix™ system 12 days later, 84 clones werepicked and transferred to 96-well plates (without selection). Singlecolonies were fed by adding 100 μl growth medium per well. Screening of96-well supernatants took place 18 days after plating. The best 24growing clones were reset to 24-well plates, by transferring thecomplete cell suspension out of the corresponding 96-well into one wellof a 24-well plate (primed with 1 mL medium (without selection). Withinthree days 24-well supernatants were analyzed and 12 clones weretransferred to 6-well plates (1 mL cell suspension+2 mL fresh growthmedium including selection). The six best expressing clones wereexpanded four days later to suspension cultivation in spin tubes (5 mLworking volume) and within four days in shake flasks (20 mL workingvolume). Two subsequent passages were performed before banking. Sixclonal cell lines were banked.

The performance of six best candidates was compared in shake flasks asdescribed above. On day 8 cell densities ranged between 9.04×10⁶ and6.40×10⁶ cells/mL and viabilities were between 74.6% and 93.1%.

Cryoconservation and Testing

After multiple passages of the clonal pools (from 6 to 31), the poolswere cryopreserved in vials at 6×10⁶ cells/vial and stored in liquidnitrogen. Absence of mycoplasma for all cell lines was confirmed byusing Venor®Gem mycoplasma detection kit (Minerva Biolabs). Sterilitytests were inoculated and incubated according to the manufacturersprotocol (Heipha, Caso-Bouillon TSB). Sterility for all minipools andsupertransfected minipools were confirmed.

Scaled-Up Cultures

The cell line designated P05ST11-cp05 was selected for scale up. For a200 liter run, the following conditions and protocol were used:

Vessel XDR-200 Bioreactor pH 7.1 ± 0.2 Dissolved Oxygen 50% Temperature37° C., see shift notes below Starting Volume 100 L Inoculum Density 1e6VC/mL Base Medium SFM4CHO Supplemented w/1XHT + (8 mM) Glutamax(Gibco ®) Feed CellBoost5 (52 g/L) 16% v:v on days 0, 3, 5, 7*CellBoost5 (52 g/L) 10% v:v days 10 and 12, further if needed. Targetculture Maintain 4-4.5 g/L glucose Feed with 40% stock as required, see“Glucose/Osmolarity” below WFI As required to maintain Osm ≦410 mOsm/kg,Supplementation see “Glucose/Osmolarity” below Harvest Criteria 60%viability Cell Viability Agitation 95 RPM Gas Sparge Design (5) 0.5 mmdrilled holes in 2 um porosity disc Cell Boost ™ Feed 16% of 52 g/L ondays 0, 3, 5, 7 10% of 52 g/L on day 10, 12, and further if neededGlucose/ measurement protocol: Feed - Measure Glucose - Osmolarity AddGlucose as Necessary - Measure Osmolarity - Add Water as NecessaryGlucose Criteria: 4-4.5 g/L Osmolarity Criteria: If >410 mOsm, add H₂0to target 390 Glutamax/ Monitor Glutamine - if drops to <0.5 mM,Glutamine supplement to 2 mM Temperature Shift Shift to 34 C. at 80% or12 × 10⁶ cells/ml Harvest Criteria Viability <60%

The expression of rhPRG4 increases in tandem with the viable celldensity (VCD) from day 1 to 8 in a 200 liter culture. The VCD plateausby day 8 then begins to fall, which is typically seen once conditionsare no longer optimal for the metabolic demands of a dense cell culturesystem. In spite of this, the expression of rhPRG4 continues unabatedand its expression in the culture system with VCD of 12-14×10⁶ cell/mlreached a maximal concentration on culture day 13. FIG. 5 shows thecumulative amount of recombinant lubricin over time as measured usingthe area under the curve of an HPLC plot, and interpreting this area bycomparison with three different standard curves made by HPLCpurification of serially diluted samples of what is believed to be atleast 99% pure lubricin. As illustrated, this procedure estimatedrecombinant lubricin production near 2.5 g/ml. Additional productionruns varied in their apparent yield as measured by various techniques.One run produced lubricin at a 1.5 g/liter level as measured bycompetitive ELISA. Another produced a reading of 1.4 g/liter.

Purification of Recombinant PRG4

The goal of development of the purification protocol is to retain thelubricating function of the expressed lubricin product and itsmultimeric complexes while separating it from contaminants, avoidingaggregation, and maintaining a high yield. This was a challenge becauseof the heavy glycosylation of lubricin, its high molecular weight, itsproperty of anti-adhesion and surface lubrication, and its tendency toform complexes, and to aggregate to form insoluble microparticles aspurity increases. Early experiments suggested that because the lubricintiter in the harvested media was high, flow through mode chromatographymight be necessary to avoid purification losses. A strategy wasdeveloped to extract contaminants by chromatographic adsorption whileretaining lubricin product in the flow through. During the course ofdevelopment it was discovered that yield was sensitive to the use ofnonionic surfactant components such as, for example, polyoxyethylenederivative of sorbitan monolaurate. Omission of such a surfactant in thelubricin pool resulted in significant loss of product during theultrafiltration/diafiltration and 0.2 μm filtration after thechromatographic separation steps. Use of as little as 0.1% by weightsurfactant greatly improved yield. By trial and error it was discoveredthat lower concentrations of surfactant succeeded in retaining functionand improving yield.

In addition to nonionic surfactants used in the purification process,physiologically compatible forms of excipients, such as[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and/orlysine may be mixed with solutions of the lubricin of the invention andcan have beneficial effects in stabilizing solutions, e.g., to avoid orreduce aggregation of lubricin in solutions containing greater than aconcentration of 0.4 or 0.6 mg/ml.

Iterative testing resulted in development of a purification procedureset forth below.

Media clarified by sedimentation (100 mL) was diluted with 5 mL 200 mMTris, 40 mM MgCl2, pH 8.2 and mixed with 400 units of Benzonase (250units/μl, Novagen) to remove soluble polynucleotides. The solution wasmixed for four hours at room temperature, then mixed with 37.8 g urea toadjust urea concentration to 6M, and to result in 120 mL of solution. Tothis was added 1N NaOH to adjust to pH 11 and 0.01% Tween 20 (sorbitanmonolaurate, Sigma).

The post-Benzonase material was next treated using GE Q Big Beads™ anionexchange resin with pH of 11 in the presence of 6M Urea and 0.01% Tween20 run in flow through (FT) mode where the contaminants bind to theresin and the product does not. The column was first sanitized with 0.1NNaOH; then charged with 100 mM NaPO4, 1.5M NaCl, pH 7.2; andre-equilibrated with 200 mM Tris-Borate, 6M Urea, pH 10. The 30 mlvolume (XK 26×6 cm) column was then loaded with the 120 ml solution at 4ml/ml resin at a flow rate of 20 ml/min (240 cm/hr), followed by a washwith equilibration buffer—100 mM Tris-Borate, 100 mM NaCl, 6M Urea,0.01% Tween 20, pH 11. Shortly after loading, product was collectedthrough the wash (290 mL total volume) until addition of a stripsolution 0.1N NaOH+1M NaCl.

This partly purified flow-through lubricin pool was pH adjusted with 1MCitrate pH=7.5, and passed through a hydroxyapatite column (BioRad CHT),Column Volume—14 ml (XK 16×7 cm), Column Load—21 ml Load/ml resin, Flowrate=10 ml/min (300 cm/hr). The column was first sanitized with 0.1NNaOH and 1 M NaCl, Charge with 500 mM NaPO₄, pH 6.5; re-equilibrationwith 500 mM NaPO₄/6M Urea, pH 7.4; and loaded with the 290 mL flowthrough from the step above. This was followed by wash withequilibration buffer, 15 mM NaPO₄, 6M Urea, 0.01% Tween 20, pH 7.4, toproduce 305 ml of flow-through containing the product.

The flow through from the hydroxyapatite column was adjusted to pH 4.8with 1M citrate and diluted with water, then passed through a GE SP BigBead resin, Column Volume—6 ml (XK 1.6×3 cm), Column Load—58 ml Load/mlresin, Flow rate=6.7 ml/min (200 cm/hr). The column was first sanitizedwith 0.5N NaOH, charge with 100 mM NaPO4, 1.5M NaCl, pH 7.4;re-equilibration with 50 mM Na citrate/6M urea, 0.01% Tween 20, pH 4.8;and loaded with the 350 mL flow through from the step above. This wasfollowed by wash with equilibration buffer, 50 mM Na citrate/6M Urea,0.01% Tween 20, pH 4.8, to produce 378 ml of flow-through containing theproduct. The flow-through was then neutralized with 10N NaOH (pH 7.2).

To concentrate and buffer exchange, the post cationic exchangeflow-through product pool was filtered using a 50 kDa molecular weightcut-off TangenX 0.01 m² flat sheet membrane (TangenX TechnologyCorporation), LP screen channel. The diafiltration buffer was 10 mMNaPO₄, 150 mM NaCl, pH 7.2 (PBS) and 0.1% Tween 20. After sanitizationwith 0.1N NaOH; a rinsed with MilliQ water; and equilibration with 10 mMNaPO4, 150 mM NaCl, pH 7.2, the membrane was loaded at 15,000 ml/m²;Cross-flow 70 ml/min; transmembrane pressure=6-7 psi; permeate flow=5-6ml/min to concentrate the solution to approximately 50 ml.

Lastly, the post UFDF product pool was subject to 0.2 μm filtrationthrough a Sartorius Sartopore 2, 150-0.015 m² membrane at a membraneload of ˜17,000 ml/m², and a flow rate of 45-50 ml/min. The membrane wasfirst primed with 10 mM NaPO4, 150 mM NaCl, pH 7.4, then the product wasfiltered, followed by a chase filter with ˜40 ml of buffer and finallythe filter was drained.

Additional excipients are currently being examined to improve recoveryfrom the UFDF and 0.2 um filtration of the final purified product. Thisprocedure can yield large amounts of product per liter of harvestedmedia of at least 96% purity. Alternative purification strategies willbe apparent to those of skill in the art.

Characterization of Lubricin Product Electrophoresis

The molecular weight of the full length lubricin amino acid backbone is150,918 Daltons. The extent and type of glycosylation varies frommolecule to molecule. The recombinant PRG4 made as disclosed herein as adimeric species is believed to have an average molecular weight ofgreater than about 450 kDa. Monomers should have a weight of 220-280kDa, and no greater than about 300 kDa.

FIG. 3 depicts a Coomassie Stained gel (Tris-Ac 3-8% NuPAGE® SDS-PAGEpolyacrylamide gel electrophoresis system, Invitrogen) of rhPRG4, bothnon-reduced as purified and reduced and alkylated. All numbered bandswere confirmed as lubricin by MS/MS, having amino acid sequence matchinghomo sapien PRG4 (UniProt Accession No. Q92954; SEQ ID NO:1). Asillustrated, recombinant lubricin produced as described above (NR)contains a major bands having approximate molecular weights, asestimated by comparison to molecular weight standards, of ˜460 kDa, oneslightly above it, and one at the top of the gel that was unable tomigrate into the gel.

Identification of post-translational processing constituents was done bydigestion of rhPRG4 with neuraminidase (NaNase 1) and O-glycosidase DSsimultaneously to expose the molecular weight of the amino acid core ofrhPRG4 as shown in the lane labeled L-NO in FIG. 4. The predictedmolecular weight of the core is 151 kDa which is experimentallyconfirmed by this 4-12% SDS-PAGE. Digestion with neuraminidase alone hada lowering effect on molecular weight illustrating that theglycosylation is incompletely capped by neuraminic acid. Digestion withO-glycosidase DS which removes O-linked β(1-3) GalNAc-Gal residues andneuraminidase implies that this batch of protein is roughly 30% byweight glycosylated. Digestion with O-glycanase alone is likely onlyeffective in removing some uncapped GalNAC-Gal residues.

Glycosylation Analysis

To further characterize the protein, mass spectrometric analysis of theO-glycans from recombinant lubricin and normal synovial lubricin wasconducted and compared. Briefly, synovial lubricin was isolated fromsynovial fluid using DEAE chromatography. Recombinant and synoviallubricin were separated by SDS-PAGE using 3-8% Tris-acetate gels beforetransferring to PVDF membrane. O-glycans were then released from thelubricin blots by reductive β-elimination followed by clean-up forLC-MS/MS analysis. O-glycans were separated by porous graphitized carbonchromatography before MS/MS analysis in negative mode using adata-dependent method on a linear ion trap mass spectrometer, LTQ(Thermo Scientific).

Analysis of the recombinant lubricin sample identified only core 1O-glycan structures (FIG. 6). The extracted ion chromatograph displayingthe identified glycans is shown in FIG. 7. The sialylated structure,[M-H]-675, is shown as two major peaks. These are the same isomer withthe second peak at retention time 21.4 min being a chemical derivativecreated during the β-elimination process. Three isomers of the sulfatedstructure ([M-H]-464) were identified. Several isomers of themonosulfated monosialylated structure also were identified. Thedisialylated structure was of very low abundance and cannot be observedin the chromatograph. An estimation of the proportion of each of theglycans identified is shown in Table 1 (for key to sugar structures, seeFIG. 6). This analysis combines all isomers, derivatives and adducts foreach of the structures in Table 1.

TABLE 1 The percentage of each of the glycans identified on therecombinant lubricin. The data includes all isomers, derivatives andadducts for each of the structures listed in the table.        

   

       

   

Glycan 384 464 675 755 966 Percentage 19.8 34.3 33.2 12.1 0.6

Normal human synovial lubricin has a larger range of glycans extendinginto the core 2 structures (FIG. 8). The most abundant of thesestructures are shown in FIG. 9 on the extracted ion chromatograph.

The glycosylation pattern of the rhlubricin is very different from thenative human glycoprotein, as can be readily appreciated, for example,from a comparison of FIG. 7 with FIG. 9. On native synovial lubricin,the sialylated core 1 structure is the most abundant glycan, but thereis significant core 2 glycosylation of various kinds, and only minoramounts of sulfated polysaccharides. On the recombinant glycoprotein,sialylated and unmodified core 1 makes up over half of the glycans, andthe sulfated core 1 structure makes up about one third of the identifiedO-glycans, with all three possible isomers identified.

Physicochemical Properties of rhlubricin Surfactant-Like (Amphipathic)Properties

An important attribute of rhPRG4 is its ability to coat both biologicaland non-biological surfaces via physicochemical adsorption. Native PRG4is surface active, and incorporates terminal globular domains separatedby the large mucin-like domain. These can separate into polar andnon-polar domains within its structure. The central mucin domain, asshown by surface force apparatus studies of human synovial fluidlubricin, can fold back upon itself suggesting that the glycosylationsare directed away as this orientation is achieved. Overall, the mucindomain becomes more hydrophilic than either its N- or C-termini. Theimportance of this is confirmed by the knowledge that digestion of theglycosylations will remove lubricating ability (Jay et al., J Glycobiol2001). This amphipathic nature also is present in rhPRG4. It can bemeasured readily by assessment of a reduction in interfacial tensionbetween an aliphatic and aqueous interface.

In an experiment designed to test the surfactant properties ofrhlubricin made using the process of the invention, an increasingconcentration of rhPRG4 was presented in a solution of PBS which wascovered by undiluted, hydrophobic cyclohexane. A Du Noüy ring placed inthe aqueous sub-phase containing rhPRG4 was pulled upward and thecritical tension ({acute over (Γ)}_(i)) where the ring breaks throughthe interface was recorded. Measurements were collected five times ateach concentration in an Attension Sigma 702ET tensiometer. A doseresponse curve of concentration of rhPRG4 was plotted against {acuteover (Γ)} i, see FIG. 10A. As illustrated, as the concentration ofrhPRG4 increases in the aqueous sub-phase containing PBS, interfacialtension decreases.

Because the rhlubricin solution contained residual nonionic surfactant(Tween 20), the experiment was repeated to investigate whether this wasresponsible for the dramatic reduction in surface tension induced byaddition of the recombinant product, first using various concentrationsof the surfactant alone, and then with very low concentrations of therhPRG4 of the invention. Microliter quantities of the surfactant andPRG4 were added to 15 mL of the aqueous sub-phase. The results are shownin FIG. 10B and FIG. 10C. As illustrated, PRG4 alone (FIG. 10C) reducessurface tension better than the commercial surfactant alone at 0.1%(FIG. 10B). Thus, rhPRG4 containing 0.1% Tween and not containing Tweenreduced interfacial tension of PBS and cyclohexane more than 0.1% Tweenalone when all had the same amounts of the solution of interest added.

These data show that even at low concentrations, rhPRG4 preferentiallypopulates the aqueous-aliphatic interface, reducing interfacial tension.This phenomenon recapitulates the surface binding interaction which isrequired in the reduction of friction and mimics the behavior of nativelubricin. Furthermore, the activity of interfacial tension reduction canbe used as a quality control procedures of rhPRG4 production.

Lubricating Properties Cartilage Lubrication

Fresh osteochondral samples (n=16) were prepared for friction testingfrom the patella-femoral groove of skeletally mature bovine stiflejoints, as described previously. Briefly, cores (radius=6 mm) andannuluses (outside radius=3.2 mm and inside radius=1.5 mm) wereharvested from osteochondral blocks, both with central holes (radius=0.5mm) to enable fluid depressurization. Samples were rinsed vigorouslyovernight in PBS at 4° C. to rid the articular surface of residualsynovial fluid, and this was confirmed by testing for the presence oflubrication. Samples then were frozen in PBS with proteinase inhibitorsat −80° C., thawed, and re-shaken overnight in PBS to further depletethe surface of any residual PRG4 at the surface. Samples were thencompletely immersed in about 0.3 ml of the respective test lubricants(described below) at 4° C. overnight prior to the next day's lubricationtest, and were again rinsed with PBS after each test before incubationin the next test lubricant.

A Bose Electroforce® test instrument (ELF 3200, Eden Prairie, Minn.) wasused to analyze the boundary lubrication ability of each of the PRG4forms and controls, using an established cartilage-on-cartilage frictiontest. Briefly, all samples were compressed at a constant rate of 0.002mm/s to 18% of the total cartilage thickness, and were allowed tostress-relax for 40 minutes to enable depressurization of theinterstitial fluid. The samples then were rotated at an effectivevelocity known to maintain boundary mode lubrication at a depressurizedcartilage-cartilage interface (0.3 mm/s) at ±2 revolutions. After beingleft in a pre-sliding stationary period of 1200, 120, 12 and 1.2seconds, samples were rotated after each subsequent stationary period,+/−2 revolutions. The test sequence was then repeated in the oppositedirection of rotation, −/+2 revolutions.

Two test sequences assessed the cartilage boundary lubricating abilityof rhPRG4, both alone and in combination with HA. In both testsequences, PBS served as the negative control lubricant and bovinesynovial fluid as a positive control lubricant. Both rhPRG4 and purifiednative bovine PRG4 were prepared in PBS at a concentration of 450 μg/mL,and HA (1.5MDA Lifecore Biomedical, Chaska, Minn.) was also prepared inPBS at a physiological concentration of 3.33 mg/mL. Lubricants weretested in presumed increasing order of lubricating ability (decreasingcoefficient of friction). In test sequence 1, rhPRG4 vs. nbPRG4, thesequence was PBS, rhPRG4, nbPRG4, synovial fluid (n=7); in test sequence2, rhPRG4 vs. rhPRG4+HA, the sequence was PBS, rhPRG4, rhPRG4+HA,synovial fluid (n=4).

The two coefficients of friction; static (μ_(static), N_(eq))(resistance of start-up motion from static condition) and kinetic(<μ_(kinetic,) N_(eq)>) (resistance of steady sliding motion) werecalculated for each lubricant as described previously. The results areshown in FIGS. 11 and 12. Data is presented as mean±SEM. ANOVA was usedto assess the effect of lubricant and pre-sliding stationary period as arepeated factor on μ_(static),N_(eq) and <μ_(kinetic),N_(eq)>, withTukey post hoc testing on <μ_(kinetic),N_(eq)> at a pre-slidingstationary period of 1.2 s. Statistical analysis was implemented withSystat12 (Systat Software, Inc., Richmond, Calif.).

As shown in FIG. 11, there was no statistical significance between themeasured lubricating property, kinetic coefficients of friction, ofrecombinant PRG4 and the slightly lower values of native bovine PRG4. Asshown in FIG. 12, rhPRG4 in combination with HA improves both static(FIG. 12A) and kinetic (FIG. 12B) lubricity as compared with rhPRG4alone. All measurements were highest in PBS and lowest in bovinesynovial fluid, with rhPRG4 and rhPRG4+HA being intermediate. The mixedsolution of rhPRG4+HA had a trend toward significantly lower coefficientof friction than rhPRG4 alone (p=0.075) and was statistically similar tobovine synovial fluid (0.021±0.001, p=0.20).

Efforts also have been made to assure removal of native lubricin frombovine cartilage intended to be used as bearings using a two-hourenzymatic digestion with hyaluronidase. Hyaluronidase digestion isintended to remove native PRG4 (P<0.050) from the superficial zone ofthe cartilage explants. This treatment removes surface PRG4 withoutsignificantly affecting the mechanical characteristics of the articularcartilage. Applying rhPRG4 to these surfaces and comparing thefrictional response to BSF and PBS controls shows that a low COF can bere-established with the rhPRG4 of the invention. FIG. 13 shows COFvalues for hyaluronidase-treated bovine medial condyle cartilageexplants with rhPRG4, BSF and PBS as intervening lubricants.Osteochondral explants were tested following the aforementionedlubricants following the protocol discussed above. As shown, recombinanthuman like PRG4 re-established a low COF (rhPRG4 N=18; BSF N=6; PBS(N=8).

Ocular Surface Lubrication

Normal human corneas with 3 mm of sclera were obtained from the SouthernAlberta Lions Eye Bank. Human eyelids were harvested from fresh cadaversfrom the University of Calgary body donation program. Approval for useand appropriation of these tissues was obtained from a Health ResearchEthics Board. The corneas (n=6) were stored in chondroitin sulfate-basedcorneal storage media (Optisol-GS) at 4° C. and used within 2 weeks. Theeyelids (n=6) were frozen and thawed at time of use.

The purity of the rhPRG4 species was assessed to be 50% by 3-8%Tris-Acetate NUPAGE sodium dodecyl sulfate polyacrylamide gelelectrophoresis. The concentration of the enriched rhPRG4 preparationwas assayed and adjusted to take the level of purity into account.

Tissue samples were mounted on a Bose ELF3200 with axial and rotationalactuators, and axial load and torque sensors. The resected cornea wasfixed to the end of a semi-spherical silicone rubber plug (radius=6 mm)by applying cyanoacrylate adhesive (superglue) to the sclera. A siliconerubber sleeve was fitted around the cornea-plug apparatus, which servedto hold lubricant fluid. This apparatus was then attached to therotational actuator of the Bose ELF3200 thus forming the bottomarticulating surface. An annulus (outer radius=3.2 mm, inner radius=1.5mm) was punched from the model PDMS material (˜0.4 mm thick UntrSylgard184, Dow Corning,) or human eyelid tissue and glued to an annulusholder. This annulus holder was then attached to the linear actuator,thus forming the upper articulating surface.

After mounting the samples, 0.3 ml of test lubricant was placed on thecornea to form a lubricant bath and the articulating surfaces wereallowed to equilibrate with the test lubricant for a minimum of fiveminutes. The tissue samples are brought into contact at three manuallydetermined axial positions to correspond with axial loads of 0.3±0.02,0.5±0.03, and 0.7±0.03 N, resulting in axial pressures ranging from 12.2to 28.5 kPa based on a contact area of (24.6 mm²). Once in contact at agiven axial position, the samples underwent four revolutions in bothdirections at four different effective sliding velocities (v_(eff)=30,10, 1.0, 0.3 mm/s) where v_(eff)=ω·r_(eff) andr_(eff)=2/3[(r_(o)3−r_(i)3)/(r_(o)2−r_(i)2)]. Axial load and torque werecollected at 20 Hz during rotations. There was a 12 second dwell timebetween each revolution. Each test sequence, described below, included apreconditioning step where the tissues underwent the described testprotocol in a saline bath.

To determine the boundary lubricating ability of the rhPRG4 preparationat a human cornea-eyelid (Test 1) and human cornea—Polydimethylsiloxane(PDMS, Test 2) interface, the following test sequence was used: 300μg/mL PRG4 in saline, 300 μg/mL rhPRG4 in saline, then saline (SensitiveEyes Saline Plus, Bausch & Lomb).

To evaluate the effectiveness of the test lubricants at the twointerfaces, static and kinetic friction coefficients were calculated. Asillustrated in FIG. 14, both PRG4 and rhPRG4, significantly andsimilarly, reduced friction at a human cornea—PDMS interface (cf. FIGS.14C and 14D) and at cornea eyelid interfaces (FIGS. 14A and 14B).

1. A method of manufacture of a recombinant lubricin glycoproteincomprising the steps of: culturing, in a medium, Chinese hamster ovary(CHO) cells transfected with and which express the human PRG4 gene andpost translationally glycosylate the expression product for a time andunder culture conditions sufficient to produce a lubricin glycoproteincomprising at least 30% by weight glycosidic residues at a concentrationin the medium of at least 0.4 g/liter, and purifying the lubricinglycoprotein from said medium.
 2. The method of claim 1, wherein the CHOcells are CHO-M cells comprising a nucleic acid encoding the human PRG4gene.
 3. The method of claim 1, wherein the CHO cells are transfectedwith a first vector comprising a nucleic acid encoding a chromatinelement and a second vector comprising a nucleic acid encoding the humanPRG4 gene.
 4. The method of claim 3, wherein the chromatin element is aboundary element, matrix attachment region, locus control region, or auniversal chromatin opening element.
 5. The method of claim 4, whereinthe chromatin element is a matrix attachment region.
 6. The method ofclaim 1, wherein the cells are cultured for a time and under cultureconditions sufficient to produce said lubricin glycoprotein at aconcentration in the medium of at least 0.5 g/liter.
 7. The method ofclaim 1, wherein the cells are cultured for a time and under cultureconditions sufficient to produce said lubricin glycoprotein at aconcentration in the medium of at least 0.8 g/liter.
 8. The method ofclaim 1, wherein the cells are cultured for a time and under cultureconditions sufficient to produce said lubricin glycoprotein at aconcentration in the medium of at least 1.0 g/liter.
 9. (canceled) 10.The method of claim 1, wherein at least 95% by weight of glycosylationof the lubricin glycoprotein is core 1 glycosylation.
 11. The method ofclaim 1, wherein at least 99% by weight of glycosylation of the lubricinglycoprotein is core 1 glycosylation.
 12. The method of claim 1, whereinthe glycosidic residues are enriched in sulfated saccharide side chainsas compared with native human lubricin.
 13. The method of claim 1,wherein the lubricin glycoprotein comprises a multimeric proteinproducing a static coefficient of friction no greater than 150% of thestatic coefficient of friction of purified native bovine lubricin asmeasured in a cartilage on cartilage friction test.
 14. The method ofclaim 1, wherein the lubricin glycoprotein comprises a multimericprotein producing a static coefficient of friction no greater than 120%of the static coefficient of friction of purified native bovine lubricinas measured in a cartilage on cartilage friction test.
 15. The method ofclaim 1, wherein the lubricin glycoprotein comprises a multimericprotein producing a static coefficient of friction no greater than 110%of the static coefficient of friction of purified native bovine lubricinas measured in a cartilage on cartilage friction test.
 16. The method ofclaim 1, wherein the lubricin glycoprotein comprises a monomericlubricin species co-purified from said culture medium and in admixturewith a multimeric lubricin species.
 17. The method of claim 13, whereinthe lubricin glycoprotein comprises a dimeric lubricin species.
 18. Themethod of claim 1 wherein the lubricin glycoprotein comprises at leastfive disulfide-bonded or non-covalently associated individualglycosylated amino acid chains and has a molecular weight of at least1200 kDa.
 19. The method of claim 1, wherein the cells are cultured inat least 10, 50, or 100 liters of medium.
 20. The method of claim 1,wherein the lubricin glycoprotein comprises at least 35% by weightglycosidic residues.
 21. (canceled)
 22. A composition of mattercomprising: a recombinant multimeric lubricin glycoprotein expressedfrom the human PRG4 gene in a host cell culture, comprising at least 30%by weight glycosidic residues, and producing a dynamic coefficient offriction no greater than 150% of the dynamic coefficient of friction ofpurified native bovine lubricin as measured in a cartilage on cartilagefriction test.
 23. The composition of claim 22, wherein the lubricinglycoprotein is characterized as producing a dynamic coefficient offriction no greater than 120% of the dynamic coefficient of friction ofpurified native bovine lubricin as measured in a cartilage on cartilagefriction test.
 24. The composition of claim 22, wherein the lubricinglycoprotein is characterized as producing a dynamic coefficient offriction no greater than 110% of the dynamic coefficient of friction ofpurified native bovine lubricin as measured in a cartilage on cartilagefriction test.
 25. The composition of claim 22, wherein the lubricinglycoprotein comprises at least 35% by weight glycosidic residues. 26.The composition of claim 22, wherein the lubricin glycoprotein comprisesat least 40% by weight glycosidic residues.
 27. The composition of claim22, wherein at least 99% by weight of glycosylation of the lubricinglycoprotein is core 1 glycosylation.
 28. The composition of claim 22,wherein the glycosidic residues are enriched in sulfated saccharide sidechains as compared with native human lubricin.
 29. The composition ofclaim 22, further comprising a monomeric lubricin species admixed withmultimeric lubricin species.
 30. The composition of claim 22, comprisinga dimeric lubricin species.
 31. The composition of claim 22, comprisinga lubricin species comprising at least five disulfide-bonded ornon-covalently associated individual glycosylated amino acid chainshaving a molecular weight of at least 1200 kDa.
 32. The composition ofclaim 22, further comprising hyaluronic acid or a salt thereof inadmixture with said lubricin glycoprotein. 33-40. (canceled)